胚胎干细胞来源肝脏组织样三维结构的构建及其在药物评价体系中的应用研究

In this project, the previously developed high efficiency differentiation induction technique of functional hepatocytes from mouse embryonic stem cells will be applied with combination of three dimensional electrospun microfiber scaffold materials to obtain large number of homogeneous hepatic proginater/stem cells. Furthermore, a stable long term culture system for the ES cells derived hepatic stem cells will be developed under detailed analysis of hepatic stem cells microenvironment. In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells。Our previous experimental data revealed that a network structure of AFP+/Alb+/E-cadherin+ hepatocytes with sinusoidal endothelial like cells invaded in it was formed through optimizing the high efficiency three step directed differentiation method of hepatocytes from ES cells. A large number of mature liver tissue specific binuclear AFP+/Alb+ hepatocyte cells were also observed in the network structure. In this project, the previous research methods and results will be combined with the differentiation induction method of hepatocytes from bone marrow meassechymal stem cells to develop a novel co-culture and co-induction method for the promotion of cell-cell interactions. Finally, a liver tissue like three dimensional structure will be constructed as a novel drug screening platform.

本项目将以已成功开发的小鼠多能干细胞向功能性肝脏细胞高效率诱导分化技术为基础，采用微米纤维为三维支架材料，获得大量均一发育状态的多能干细胞来源肝脏祖细胞，并对肝脏祖细胞的微环境进行精细的解析，首先建立长期稳定培养肝脏祖细胞的方法。肝脏发育进程中，肝母细胞具有分化为肝脏实质细胞和胆管上皮细胞的潜能。先行的研究结果表明，通过改进多能干细胞向肝脏细胞高效率诱导的三步法，可以实现AFP+/ALB+/E-cadherin+肝脏细胞中侵入肝窦内皮细胞样的网状二维结构，并发现诱导形成的网状结构中呈现很多成熟肝脏组织特性的双核 AFP+/ALB+肝脏细胞。本项目以先行的研究结果为基础，结合骨髓来源间充质干细胞向肝脏细胞诱导分化的方法，运用共培养和诱导体系，促进细胞之间的相互作用，构建肝脏组织样三维结构，为药物筛选体系提供新的手段和技术平台。

本项目中采用静电纺丝微纤维和E-钙黏素融合蛋白质的三维支架复合材料，初步获得了发育状态均匀的多能干细胞，并运用单细胞测序技术详细分析了组成小鼠早期胚胎肝脏的细胞种类以及不同细胞的微环境的状态。单细胞RNA-Seq分析结果得出，E11.5天小鼠胚胎肝脏组织中细胞主要处于游离状态，胎肝发育中血管的侵入预先发生在肝的形成与造血发育开始之前。运用密度梯度差速离心法，从每个E11.5胚胎肝脏中平均分离出43.7×104个肝脏干/祖细胞，其中Dlk1+比率占30%以上的细胞。以Fibronectin为培养基质,在含有HGF、FGF2、FGF10细胞因子的培养体系中，分离得到的肝脏干/祖细胞可稳定培养7天以上时间。对培养过程中肝脏干/祖细胞进行分子水平鉴定，发现肝脏干/祖细胞均表达维持干性的不同标记基因，即具有分化为肝脏实质细胞和胆管上皮细胞的潜能。.综上所述，应用密度梯度差速离心法成功研发了无损伤小鼠肝脏祖细胞的分离方法；采用多种细胞外基质结合不同细胞因子初步研发出肝脏祖细胞的体外长期稳定培养体系；为胚胎干细胞向肝脏祖细胞的高效诱导及肝脏类器官的制备提供了有效理论依据；大鼠肝纤维化/肝硬化模型的研究中，临床药物藏红花-13味散的治疗效果的病理以及分子和细胞水平的检测分析取得了一定的进展。